Embodiments and aspects of the invention are generally directed to methods and associated apparatus for automatically aligning an aperture in an optical system and applications thereof. More particular embodiments and aspects are directed to methods and associated apparatus for automatically aligning one or more pin-hole-sized apertures in an optical system having multiple light sources and light detection wavelengths in one or more channels of an optical imaging system, particularly in the presence of at least certain optical aberrations such as, but not limited to, chromatic aberration. Most particularly, embodiments and aspects are directed to methods and associated apparatus for automatic alignment of a confocal aperture in a confocal imaging system such as, but not limited to, a confocal scanning laser microscope or a fluorescence adaptive optics scanning laser ophthalmoscope (FAOSLO), especially for fluorescence imaging in the human eye where the chromatic aberration of the eye under investigation is unknown. These embodiments and aspects offer, for example, a synergistic enhancement enabling high resolution imaging of individual retinal pigment epithelium (RPE) cells in patients with age-related macular degeneration (AMD).
Drusen and atrophy of the retinal pigment epithelium (RPE) are hallmarks of age related macular degeneration (AMD). Histological studies of postmortem eyes have shown that substantial changes occur in RPE cell mosaic morphology in AMD. These changes may precede and/or accompany RPE cell death and the degeneration of overlying photoreceptors. Clinical imaging methods, such as confocal scanning laser ophthalmoscopy (cSLO), are used to examine the fundus fluorescence (FAF) pattern as a means of assessing the health of the RPE in AMD and other retinal diseases. However, currently available commercial FAF imaging systems lack the resolution to identify individual cells, preventing morphometric analysis of the RPE cell mosaic.
Although imaging of the intrinsic autofluorescence (AF) of lipofuscin has become a relatively common method used in the clinic to assess the health and integrity of the RPE, there are several reasons why higher resolution images of the RPE would be desirable to evaluate patients with diseases affecting the RPE. FAF images obtained using commercial instruments have demonstrated utility for observing the overall patterns of RPE fluorescence seen in disease, but these images are often difficult to interpret. Sophisticated qualitative classification schemes have been proposed but are difficult to implement without trained readers and are limited in their utility. The total absence of an FAF signal is often interpreted as complete RPE atrophy and FAF images can be useful for assessing the extent of RPE loss in geographic atrophy (GA) and its progression rate. However, spectral domain optical coherence tomography (SD-OCT) has been shown to be more precise for measuring lesion size in GA. This is due in part to the variability in fluorescence seen at the borders of GA lesions and by screening of fluorescence due to macular pigment. For these reasons and since the fluorescence signal obtained is not a radiometric measurement (i.e., the images display relative, not absolute AF), the interpretation of hyper-AF and hypo-AF patterns seen in diseased eyes is difficult. These patterns presumably relate to changes in the health and integrity of the RPE cell mosaic, but cellular resolution is needed to understand how RPE cell mosaic morphology relates to the patterns seen in FAF images. Histological studies have shown that RPE mosaic morphology is drastically altered in AMD, but these studies of post mortem eyes are limited in that they only reveal a single time point and cannot compare changes in RPE morphology to other measures, such as cSLO FAF imaging, SD-OCT, or AO reflectance imaging of the photoreceptor cell mosaic. In-vivo cellular resolution imaging has the potential to identify early disease changes in the RPE cell mosaic, before the heterogeneous patterns of hyper-AF and hypo-AF seen in conventional FAF imaging arise. Furthermore, cellular imaging could enable earlier detection of retinal disease, improved understanding of disease etiology, more rapid monitoring of disease progression, and more sensitive metrics for evaluating treatment effects.
By combining fluorescence imaging methods with adaptive optics scanning light ophthalmoscopy (FAOSLO), Gray, Morgan, and colleagues demonstrated that it was possible to image individual RPE cells in-vivo in monkeys. Morgan and colleagues later demonstrated that these methods could be used to achieve single cell resolution of the RPE in the living human eye. Since the RPE is important for maintaining the healthy function of the photoreceptor layer and is implicated in many retinal diseases, such as AMD, the demonstration that these cells were now accessible to optical imaging in the living human eye was a potentially valuable advance. However, the instant inventor's early attempts to image the RPE in patients with AMD using these methods proved difficult; images with greater structural detail than provided by commercial systems were obtained, but individual cells could not be resolved.
This difficulty can be partially attributed to the aging eye, which poses a challenge for imaging even using adaptive optics ophthalmic imaging instruments. Optical challenges that affect image contrast and resolution include increased scatter, lens opacities, and dry eye. Patients with AMD whose central vision is compromised usually have poor fixation, which can increase distortions in scanning system images and make image registration difficult. Moreover, older adults can often have other health problems or mobility issues requiring imaging sessions to be short. All of these factors conspire to make imaging the aging eye more difficult than for younger eyes. Despite having success imaging RPE cells in some healthy young eyes using the fixed dual focus method proposed by Morgan and colleagues, our ability to resolve the RPE mosaic was highly inconsistent. This was due in part to poor compensation of the longitudinal chromatic aberration of the eye. This procedure proved difficult to replicate reliably using manual positioning of the optical elements, such as lenses, light sources, and confocal aperture(s). This is supported by early experiments that suggested that a fixed defocus offset to compensate for chromatic aberration did not appear to work consistently for all observers. This inconsistency was due to a combination of optical alignment and true differences in longitudinal chromatic aberration between participants.
The current standard method for adjusting the focal plane of an AOSLO is to use the deformable minor to change the focus. This works well when one light source is being used to image the retina; however, when multiple wavelengths of light are used to image different layers of the retina simultaneously, this method is not ideal. This is due to the fact that all of the light sources will change focus together when adjusted using the deformable mirror. Since changes in the focus between retinal layers arise due to location or retinal pathology, it is very difficult to simultaneously maintain the focus of each wavelength on the retinal layer of interest, or to change the focus so that multiple wavelengths could simultaneously be focused on different retinal layers or on the same layer depending upon experimental necessity. The focus of the light can be altered for each light source independently by altering the vergence of the light before entering the optical system. However, for a given defocus, the confocal aperture and PMT detector position must also be changed (in three dimensions) to be placed in the proper position for light detection from the retina. Furthermore, when the focus is changed in this way it alters the amount of light that is coupled into the imaging system. This is an important consideration for imaging the living human eye, as one could increase the fluorescence simply by increasing the power of the excitation light by coupling more light into the system. By altering the focus and the power simultaneously, and monitoring the power of the emitted light to determine the appropriate focus, it remains unknown whether the increase in emission is due to obtaining the appropriate focus or due to simply altering the power of the excitation light. Moreover, visible light can be harmful to the retina and it is important to know how much light one is putting into the eye for safety reasons. If one adjusted the focus of the ingoing light while imaging the eye without simultaneously measuring and adjusting the power of the ingoing light, the retina may be damaged. Accordingly, apparatus and techniques known in the art may be employed for limiting visible light exposure; for example, a shutter under computer control may be used to limit light exposure while the common focusing element and the pinhole translation stage are being manipulated to their desired focus/positions. Alternatively, the source may be electronically modulated. Other approaches are known in the art.
In view of the foregoing, the inventor has recognized the benefits and advantages in providing an enabling solution to address these problems in the form of a method and system for automatically aligning an aperture in an optical system; more particularly, for automatically aligning one or more pin-hole-sized apertures in an optical system having multiple light sources and light detection wavelengths in one or more channels of an optical imaging system, particularly in the presence of at least certain optical aberrations such as, but not limited to, chromatic aberration; and most particularly, for automatic alignment of a confocal aperture in a confocal imaging system such as, but not limited to, a confocal scanning laser microscope or a fluorescence adaptive optics scanning laser ophthalmoscope (FAOSLO), especially for fluorescence imaging in the human eye where the chromatic aberration of the eye under investigation is unknown. The embodied invention enables one to minimize light exposure to the eye and optimize fluorescence excitation and emission detection without altering the amount of light that is put into the eye.